The discussion of any work, publications, sales, or activity anywhere in this submission, including in any documents submitted with this application, shall not be taken as an admission that any such work constitutes prior art. The discussion of any activity, work, or publication herein is not an admission that such activity, work, or publication existed or was known in any particular jurisdiction.
Handling, characterization, and visualization of individual cells has become increasingly valued in the fields of drug discovery, disease diagnoses and analysis, and a variety of other therapeutic and experimental work. However, few high-resolution methods exist to control and manipulate the biochemical nature of a single cell's interior; yet roughly 90% of the cell's biologically active structures, such as intracellular proteins, are located within the confines of the cell membrane. The cell membrane serves as an effective barrier between the cytoplasm and the outside world and, as such, is relatively impermeable to most ionic and polar substances.
One way to and access the cell's interior is by temporarily increasing the cell membrane's permeability. This can be accomplished via electroporation, a technique using high electric fields to induce structural rearrangements of the cell membrane. Pores are formed when the transmembrane potential exceeds the dielectric break-down voltage of the membrane (about 0.2-1.5V). Material such as polar substances other-wise impermeant to the plasma membrane (such as dyes, drugs, DNA, proteins, peptides, and amino acids) can thus be introduced into the cell.
In the early 1980s, Eberhard Neumann et al. demonstrated the feasibility of electroporation for delivering DNA to a population of mammalian cells. Since then, methods of bulk electroporation has become a standard technique routinely used to simultaneously transfect millions of cells in culture. However, bulk electroporation requires very high voltages (>103V) and has little control over the permeabilization of individual cells, resulting in suboptimal parameters. Reversible electroporation, in which the pores can reseal, is therefore difficult.
While single cell electroporation has been hoped to have advantages over bulk methods, practical systems or methods for single cell electroporation have generally been less commonly used than bulk methods. Lundqvist et al. first demonstrated single cell electroporation using carbon fiber microelectrodes in 1998. To induce electroporation, they placed the microelectrodes 2-5 microns away from adherent progenitor cells. Other single cell electroporation techniques developed since include: electrolyte-filled capillaries, micropipettes, and microfabricated chips.
In 2001, Huang et al. introduced a microfabricated single cell electroporation chip. In 2002, Nolkrantz et al. demonstrated functional screening of intracellular proteins by using electroporation to introduce fluorogenic enzyme substrates and receptor ligands into single cells.
Chip-based cellular handling devices have been proposed using silicon oxide coated nitride membranes, silicon elastomers, polyimide films, quartz or glass substrates. Recently, three dimensional structures more similar to patch pipettes have also been fabricated. Some earlier chip-based devices developed to date generally use a horizontal geometry where the patch pore is etched in a horizontal membrane dividing the top cell compartment from the recording electrode compartment.
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